This application seeks funding for studies designed to dissect T cell responses to the capsid antigens of adeno-associated viruses (AAV), different types of AAV vectors and transgene products of such vectors. The goal of this renewal application is to further our understanding on detrimental immune responses that arise upon AAV gene transfer and interfere with sustained expression of therapeutic proteins. In aim 1 we will determine how long CDS"^ T cells recognize capsid epitopes of different AAV vectors using methods developed during the current funding period. In aim 2 we will characterize CD8* T cell responses to the transgene product of double-stranded (ds)AAV vectors. As we showed during the last funding period, transgene product-specific CD8'^ T cells induced in mice by single-stranded (ss)AAV vectors are functionally Impaired. In more recent studies we observed that dsAAV vectors stimulate by far more potent CDS* T cell responses at lower doses, which appear to be functional unlike those Induced by ssAAV vectors. We will conduct additional studies to assess immune responses to transgene products of dsAAV vectors and to elucidate if and to what degree such responses interfer with sustained gene transfer. In aim 3 we will characterize T cell responses to natural AAV infections in non-human primates (NHPs) and in humans. As we showed during the current funding period, both humans and NHPs have memory T cells to AAV capsid. Frequencies of such T cells are very robust in NHPs and markedly lower in humans. This initially appears to contradict the basic hypothesis of this program that pre-existing memory CDS* T cells to AAV capsid are reactivated upon AAV-mediated gene transfer and then cause rejection of cells transduced with AAV vectors expressing a therapeutic protein, as this potential problem was only encountered in humans but not In NHPs. Our hypothesis is that AAV capsid-specific CDS* T cells from humans and NHPs differ in their functionality and that those from humans can mount an effective recall response, while those from rhesus macaques are unable to do so, as will be investigated In depth using samples from NHPs (aim 3.1.), healthy human adults (aim 3.2.1.) and children convalescent from an infection with an adenovirus and potentially AAV (aim 3.2.2). An additional puzzling finding from a clinical AAV gene transfer patient will be followed up in aim 3.2.3. In this trial a patient was treated by hepatic infusion of an AAV2-hF.IX vector while under Immunosuppression (IS). He developed an increase In circulating AAV capsid-specific T cells following gene transfer without showing evidence of liver cell destruction. The pathways underlying this increase of apparently dysfunctional AAV capsid-specific CDS* T cells will be elucidated further with samples from transplant patients taken before and/after initiation of IS.